WO2021209080A2 - Peptide for the treatment of cytokine storm syndrome - Google Patents

Peptide for the treatment of cytokine storm syndrome Download PDF

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Publication number
WO2021209080A2
WO2021209080A2 PCT/CU2021/050001 CU2021050001W WO2021209080A2 WO 2021209080 A2 WO2021209080 A2 WO 2021209080A2 CU 2021050001 W CU2021050001 W CU 2021050001W WO 2021209080 A2 WO2021209080 A2 WO 2021209080A2
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Prior art keywords
syndrome
seq
pharmaceutical composition
peptide
treatment
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PCT/CU2021/050001
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Spanish (es)
French (fr)
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WO2021209080A3 (en
Inventor
María del Carmen Domínguez Horta
Rafael VENEGAS RODRIGUEZ
Gillian Martinez Donato
Gerardo Enrique Guillen Nieto
Mabel HERNÁNDEZ CEDEÑO
Lilia María ORTEGA GONZÁLEZ
Hugo NODARSE CUNI
Cruz Matilde Lopez Abad
Hilda Elisa Garay Perez
Raimundo UBIETA GÓMEZ
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Centro De Ingenieria Genetica Y Biotecnologia
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Priority to KR1020227036582A priority Critical patent/KR20220167289A/en
Priority to AU2021256814A priority patent/AU2021256814A1/en
Priority to CN202180027963.9A priority patent/CN115461069A/en
Priority to CA3172251A priority patent/CA3172251A1/en
Priority to BR112022020442A priority patent/BR112022020442A2/en
Priority to US17/918,299 priority patent/US20230190866A1/en
Application filed by Centro De Ingenieria Genetica Y Biotecnologia filed Critical Centro De Ingenieria Genetica Y Biotecnologia
Priority to EP21754903.9A priority patent/EP4137144A2/en
Priority to JP2022559505A priority patent/JP2023521592A/en
Priority to MX2022012809A priority patent/MX2022012809A/en
Publication of WO2021209080A2 publication Critical patent/WO2021209080A2/en
Publication of WO2021209080A3 publication Critical patent/WO2021209080A3/en
Priority to ZA2022/10315A priority patent/ZA202210315B/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the branch of biomedicine and the pharmaceutical industry, in particular with a pharmaceutical composition for the treatment and prevention of cytokine storm syndrome.
  • Said composition comprises the peptide identified as SEQ ID NO: 1, and can be used in the treatment of acute respiratory distress syndrome, macrophage activation syndrome, respiratory sepsis, dengue, hepatic encephalopathy and pericarditis, among others. diseases.
  • the epithelial barrier is also altered, and it exacerbates an inflammatory response with secretion of pro-inflammatory IL, such as TNFa, IL-1b, and IL-6, and other mediators of inflammation, with the successive activation of neutrophils, monocytes, macrophages, and the trafficking of these molecules and cells towards the alveolar spaces, increasing the initial involvement.
  • pro-inflammatory IL such as TNFa, IL-1b, and IL-6
  • IL-6 pro-inflammatory IL-6
  • the development of this hyperinflammation occurs in other diseases such as respiratory sepsis, macrophage activation syndrome, and sepsis in adults and neonates.
  • the present invention solves the problem raised above, by providing a pharmaceutical composition for the treatment and prevention of cytokine storm syndrome that comprises a concentration of 1.8-3.6 mg / mL of the peptide identified as SEQ ID NO: 1 and a sucrose concentration between 20-30 mg / mL, which surprisingly, synergistically increases the biological activity of said peptide, in addition to being a determining factor for the induction of regulatory T cells, which control the magnitude of the inflammation.
  • the composition of the invention is capable of reducing the hyperinflammation that characterizes the subgroup of patients with Covid-19 who transition to a severe stage, characterized by respiratory distress, as occurs in respiratory sepsis, macrophage activation syndrome, and neonatal sepsis and adult.
  • Administration of the peptide identified as SEQ ID NO: 1 reduces the level of pro-inflammatory cytokines in patients with Covid-19 disease, without causing immunosuppression or serious adverse effects or those associated with the peptide therapy.
  • the peptide identified as SEQ ID NO: 1 is derived from a region of the HSP60 protein, comprised between amino acids 83 to 109.
  • HSP60 is highly immunogenic, both in healthy individuals and in patients with autoimmune diseases. However, the level of antibodies and T cells that this protein can induce is higher in patients with autoimmune diseases (de Jong et al ⁇ 2009). Arthritis Rheum. 7 (60): 1966-1976). This protein has a great capacity to stimulate the pro-inflammatory reactivity of the innate immune system. HSP60 has also been found to induce the expression of IL-12 and IL-15 genes, cytokines involved in the induction of a Th1 phenotype.
  • HSP60 acts as a danger signal for the innate immune system (Habich et al ⁇ 2005). J.lmmunol. 3 (174): 1298-1305). On the other hand, peptides derived from HSP60 can also constitute a danger signal (Quintana and Cohén (2011). Trends Immunol. 2 (32): 89-95).
  • the peptide of SEQ ID NO: 1 contains several overlapping T-cell epitopes. It increases the frequency of Treg cells with CD4 + CD25 high Foxp3 + phenotype in ex vivo assays with peripheral blood mononuclear cells from rheumatoid arthritis patients. However, this is not the case with cells from healthy donors (Barbera et al (2016). Cell Stress. Chaperones 4 (21): 735-744).
  • the peptide identified as SEQ ID NO: 1 is obtained by chemical synthesis. Through experiments such as those described in the present invention, those immunological mechanisms induced by the peptide that they participate in the control of hyperinflammation. The pharmaceutical composition was well tolerated and with a high safety profile, with no evidence of immunosuppression. No serious adverse events were identified in any patient.
  • cytokine storm syndrome is understood to be the increase in pro-inflammatory cytokines, where concentrations that exceed normal levels are reached.
  • pro-inflammatory cytokines are IL-6, TNFa, IL-1, IL-17, among others. It also refers to the increase in other cytokines, such as IL-10, IL-8, and IL-5.
  • biomarkers of inflammation such as C-reactive protein, fibrinogen, D-dimer, liver transaminases, lactate dehydrogenase and ferritin.
  • the increase in these molecules associated with the cytokine storm occurs in patients affected by inflammatory pathologies such as: acute respiratory distress, macrophage activation syndrome, respiratory sepsis, dengue, hepatic encephalopathy and pericarditis. From the clinical point of view, sustained fever, weakness, fatigue and nausea can be evidenced in patients.
  • the invention discloses the use of a pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1 in the manufacture of a medicament for the treatment and prevention of cytokine storm syndrome.
  • treatment with a composition comprising said peptide allows the withdrawal of mechanical ventilation in patients with Covid-19, from the improvement of interstitial pneumonia, the reduction of reactants of the acute phase of inflammation among 24 and 48 hours, and the reduction of biomarkers of inflammation.
  • the rapid decrease in C-reactive protein, in just 24 hours, is an unexpected finding.
  • Previously, in patients with rheumatoid arthritis who were treated with this peptide it was not possible to demonstrate a significant decrease in C-reactive protein (Prada, et al (2016). J Clin Triáis 1 (8): 1-11).
  • the present invention demonstrates the ability of the peptide identified as SEQ ID NO: 1, administered intravenously, to reduce respiratory distress, hyperinflammation, the reactants of the acute phase of inflammation, and the level of pro-inflammatory cytokines in the patients with Covid-19. Surprisingly, these results were reproduced in patients with respiratory sepsis who presented with the respiratory distress syndrome.
  • the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1 is used in the manufacture of a drug for the treatment and prevention of cytokine storm syndrome that manifests as acute respiratory distress syndrome, macrophage activation syndrome, respiratory sepsis in adults or neonates, systemic inflammatory response syndrome, venous or arterial thromboembolism, acute elevation of ferritin levels, severe pericarditis, acute elevation of liver transaminases causing hepatic encephalopathy or disease associated with therapies with T cells that possess chimeric antigen receptor.
  • cytokine storm syndrome that manifests as acute respiratory distress syndrome, macrophage activation syndrome, respiratory sepsis in adults or neonates, systemic inflammatory response syndrome, venous or arterial thromboembolism, acute elevation of ferritin levels, severe pericarditis, acute elevation of liver transaminases causing hepatic encephalopathy or disease associated with therapies with T cells that possess chimeric antigen receptor.
  • the cytokine storm syndrome that underlies the systemic inflammatory response syndrome, which occurs in patients after burns or severe trauma or in acute inflammatory diseases.
  • the drug is administered intravenously.
  • the medicament is used for the treatment and prevention of cytokine storm syndrome caused by an infectious disease.
  • said infectious disease is Covid-19 or dengue.
  • the object of the present invention is a method for the treatment or prevention of cytokine storm syndrome that comprises the administration of a pharmaceutically effective amount of a pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1, to an individual who does so. needs to.
  • the cytokine storm syndrome manifests as acute respiratory distress syndrome, macrophage activation syndrome, respiratory sepsis in adults or neonates, systemic inflammatory response syndrome, venous or arterial thromboembolism, acute elevation of levels.
  • the cytokine storm that is treated or prevented by the method of the invention is caused by an infectious disease.
  • the method of the invention is useful for the treatment of the infectious disease Covid-19 or dengue.
  • the composition in said method is administered intravenously.
  • composition comprising the peptide identified as SEQ ID NO: 1 to patients in need thereof reduces the events associated with respiratory distress. This distress causes patients to need:
  • composition of the invention is useful for the treatment of patients affected with the infectious disease Covid-19, when said patients have a need for oxygen therapy of no less than 6 L / min plus one of the following conditions:
  • composition of the invention is also useful for the treatment of patients with Covid-19 who enter a serious state without the need for oxygen therapy, but who present an increase in the initial value of ferritin from 500 ng / ml or value absolute ferritin greater than or equal to 2000 ng / ml, with progressive radiological signs of multifocal interstitial pneumonia.
  • FIG. 1A Frequency of T cells quantified by flow cytometry expressing the CD4 + Foxp3 + phenotype, isolated from the spleen of BALB / c mice, after being inoculated with the pharmaceutical composition of the peptide identified as SEQ ID NO: 1, where sucrose was added at a concentration 10 mg / mL. (10), 30 mg / mL (30) and 80 mg / mL (80). The Kruskal-Wallis and Dunn statistical tests were used for data analysis. Different letters indicate statistically significant differences (P ⁇ 0.05).
  • FIG. 1B Frequency of T cells quantified by flow cytometry expressing the CD4 + Foxp3 + phenotype, isolated from the spleen of BALB / c mice, after being inoculated with the pharmaceutical composition of the peptide identified as SEQ ID NO: 1, where sucrose was added at a concentration of 20 mg / mL. (20), 30 mg / mL (30) and 40 mg / mL (40). The Kruskal-Wallis and Dunn statistical tests were used for data analysis. Different letters indicate statistically significant differences (P ⁇ 0.05).
  • the pharmaceutical composition was administered intravenously, once every 12 hours. In eight of the ten patients, 1 mg of the peptide was administered per patient in each application. Patients with code 5 and 6 received 2 mg every 12 hours, as they had cancer among the comorbidities. The characteristics of the patients, as well as the days of VAM APRV and administration of the peptide are reflected in Table 1.
  • HT arterial hypertension.
  • VAM APRV Mechanical Artificial Ventilation / Airway Pressure Release Ventilation.
  • the ten patients with Covid-19 disease who were treated with the peptide had risk factors that predispose to a serious or critical state of the same. Only one patient had no comorbidities, but his advanced age (80 years) was a risk factor. In particular, the patient identified with no. 5 presented a deep vein thrombosis. However, the clinical evolution of the ten, once they were administered the peptide, was favorable. At the time of extubation, the acute respiratory distress syndrome is considered to have ended. These results demonstrate that the peptide treatment was effective for the acute respiratory distress syndrome that these patients develop.
  • Example 3 Decrease in C-reactive protein in patients with Covid-19 disease treated with the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1.
  • C-reactive protein is one of the most used markers for inflammatory processes, it is considered one of the reactants of the acute phase of inflammation.
  • the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1 was administered intravenously. Eight of the ten patients affected with Covid-19, whose treatment is described in Example 1, received 1 mg of the peptide every 12 hours. Patients with code 5 and 6 who had cancer between comorbidities received 2 mg of the peptide every 12 hours. Unexpectedly, treatment with the peptide of SEQ ID NO: 1 decreased the levels of C-reactive protein, markedly, in the treated patients. These results are shown in Table 3.
  • the decrease in C-reactive protein during the treatment of these patients is a direct indication that the peptide of SEQ ID NO: 1 reduces inflammation in them, and is in correspondence with the disappearance of the acute respiratory distress syndrome, as well as with the clinical and radiological improvement of the patients.
  • Example 4 Decrease in pro-inflammatory cytokines in patients with Covid-19 disease treated with the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1.
  • the levels of the pro-inflammatory cytokines IL-6, IL-17, IL-1, IFNy and TNFa were determined in patients with Covid-19 disease treated with the composition comprising the peptide identified as SEQ ID NO: 1.
  • the patients were in critical condition.
  • the treatment of these patients was set forth in Example 1.
  • These cytokines were quantified in the sera of the patients identified with the numbers 1, 2, 3, 4, 7 and 8, which appear in Table 1, Example 1.
  • the virus under strict biosecurity measures. The quantification was carried out just before applying the first dose of the peptide, and at 48, 72 and 96 hours after starting the treatment with it.
  • cytokine determinations were carried out using an ELISA (enzyme-linked immunosorbent assay) specific for each one, following the manufacturer's recommendations (Quantikine® R&D Systems, USA). Briefly, 50 pL of the specified diluent for each cytokine was added to the wells, followed by 200 pL of the standard curve, sample or control. The plate was incubated for 2 h at room temperature, and then the contents of each well were discarded. Then three washes were performed with the 1X wash solution, 200 pL of the conjugated antibody was added to each well, and incubated for 1 hr at room temperature. The wash was repeated and 200 pL of the substrate solution was added to each well.
  • ELISA enzyme-linked immunosorbent assay
  • the plate was incubated for 20 min at room temperature protected from light, 50 pL of the stop solution was added to each well, and the optical density was determined at 450 nm in a microplate reader (Thermo Scientific Multiskan Go, Finland) . A second reading was made at 570 nm, and the values obtained were subtracted from the 450 nm reading, to correct the optical imperfections on the plate. The determination of the concentration of each cytokine was carried out in triplicate. The results obtained for IL-6 are shown in Table 4.
  • IL-6 cytokine levels (pg / mL) found in patients treated with the peptide. As can be seen in the table, there was a decrease in the levels of the cytokine IL-6, since the critically ill patients received the peptide of SEQ ID NO: 1. This is a cytokine that increases markedly in inflammatory processes . It has been described that patients with Covid-19 disease who transition to a severe stage have elevated this cytokine, in contrast to patients who present mild symptoms of the disease. Similarly, a decrease in IL-17, IL-1, IFNy and TNFa was found in these patients. These results confirm that treatment with the peptide reduces hyperinflammation, and cytokine storm, developed by patients with Covid-19 disease who are transitioning to a severe stage.
  • Example 5 Treatment of a patient with Covid-19 disease who presented high concentrations of ferritin without respiratory distress with the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1.
  • the patient treated was 66 years old, female, with a history of type 2 diabetes mellitus, exogenous obesity, grade 4 fatty liver disease. He was admitted, for a diagnosis of COVID-19, presented a digestive picture due to dyspepsia, nausea, occasional diarrhea , for which prokinetics and anti-emetics were indicated. In addition, he presented high concentrations of ferritin, above 1500 ng / mL, and presented an increase in the erythrocyte sedimentation rate with a fall in hemoglobin. Due to this clinical picture, the specialists indicated treatment with the pharmaceutical composition that comprises the peptide identified as SEQ ID NO: 1, at a dose of 1 mg, every 12 hours.
  • the ferritin values were reduced to a concentration of 350 ng / mL.
  • Treatment with the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1 continued for a further 72 hours.
  • the ferritin concentration normalized, and the patient did not present any symptoms.
  • Example 6 Treatment of a patient with pericarditis of viral etiology with the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1.
  • the treated patient was 48 years old, confirmed with Covid-19, and admitted to the Holgu ⁇ n Military Hospital. After two days of admission, the patient presented a fever of 38C and two diarrheal stools, and was diagnosed with pericarditis secondary to myocarditis, of viral etiology (Covid-19), without hemodynamic repercussions. The patient did not present respiratory distress.
  • Treatment was started with the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1, at a dose of 1 mg every 12 hours.
  • the electrocardiogram showed improvement, and at 96 hours of treatment the patient was asymptomatic, with significant clinical improvement.
  • Example 7 Treatment of a patient with Covid-19 hepatic encephalopathy with the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1.
  • the patient was suspended all the hepatotoxic medications, changes were made to the diet, the gradual replacement of sodium was continued, and the treatment with the pharmaceutical composition that comprises the peptide identified as SEQ ID NO: 1 was started, at a rate of 1 mg every 12 hours, intravenously.
  • the patient was transferred to the intensive care unit, and the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1 was administered, at a rate of 1 mg every 12 hours, intravenously.
  • the administration of said composition had the objective of modulating the immune response, and thus controlling the cytokine storm, which, according to the clinical picture and laboratory parameters, was triggered in this patient.
  • After 48 hours of treatment a noticeable improvement was observed in the patient, since abdominal pain, arterial hypotension and distal coldness disappeared.
  • the patient began taking food by mouth.
  • clinical and radiological improvement continued, from which the frequency of administration of the pharmaceutical composition comprising the peptide was reduced to 1 mg every 24 hours.
  • the patient was clinically and radiologically asymptomatic. Dengue virus infection was confirmed, as IgM was positive in the serological test. All laboratory parameters were normalized, and the patient was discharged. These results confirmed those outlined in the previous examples, it is confirmed that the pharmaceutical composition that comprises the peptide identified as SEQ ID NO: 1 controls the cytokine storm, which can be linked to the hemophagocytic syndrome that can be associated with various pathologies, such as the case of patients with dengue who transition to a severe stage of the disease.
  • Example 9 Effects of the sucrose concentration on the pharmacological potentiation of the peptide of SEQ ID NO: 1 for the reduction of inflammation.
  • composition For the preparation of the composition, 1.8mg / mL - 3.5mg / mL of the peptide identified as SEQ ID NO: 1 were used and it was dissolved in sodium acetate buffer, pH 4; to which sucrose sugar was added in different concentrations, as described later.
  • mice of the BALB / c line were randomly separated into three groups of 10 each.
  • the mice separated by group were inoculated intravenously, as follows:
  • -Group 1 pharmaceutical composition of the peptide identified as SEQ ID NO: 1, where sucrose was added at a concentration of 10 mg / mL.
  • -Group 2 pharmaceutical composition of the peptide identified as SEQ ID NO: 1, where sucrose was added at a concentration of 30 mg / mL.
  • -Group 3 pharmaceutical composition of the peptide identified as SEQ ID NO:
  • mice received a second dose under identical conditions.
  • the 10 animals per group were sacrificed on day 8 after receiving the first inoculation.
  • the spleen cells of the mice were preincubated with Mab 2.4G2 (eBiosciences, USA), to block non-specific binding to FC receptors.
  • the cells were labeled in PBS containing three percent calf serum and saturating amounts of the following mAbs (eBiosciences, USA): APC-Cy7-anti-CD4 conjugate (clone RM4-5) and APC-anti-CD25 conjugate ( PC61).
  • the Foxp3 transcriptional factor was evaluated through the use of an anti-Foxp3 mAb conjugated to PE (FJK-16s; eBioscience, USA), following the manufacturers' recommendations.
  • An isotype-irrelevant mAb (rat PE-Cy5 IgG2a; eBioscience, USA) was used as a control.
  • the region corresponding to lymphocytes was framed according to their distribution characteristics in the Flow cytometric histograms on an LSR-II TM cytometer. The analyzes were carried out using the FlowJo® program (Tree Star).
  • mice inoculated with the formulation containing the peptide identified as SEQ ID NO: 1 and sucrose at a concentration of 30 mg / mL significantly increased the percentage of regulatory T cells, which shows a synergistic effect between those components of the composition.
  • sucrose at this concentration is decisive to induce the frequency of regulatory T cells, we carried out an experiment under conditions very similar to the previous one, but narrowing the concentration range of said sugar.
  • mice were randomly separated into three groups of 10 mice each.
  • the mice separated by group were inoculated intravenously, as follows:
  • Group 1 pharmaceutical composition of the peptide identified as SEQ ID NO: 1, where sucrose was added at a concentration of 20 mg / mL.
  • sucrose was added at a concentration of 30 mg / mL.
  • mice treated with these sucrose concentrations between 20-40 mg / mL showed a significant increase in the percentage of regulatory T cells compared to the groups treated with 10-80 mg / mL in the previous experiment, observing a synergistic effect between the components of the composition, demonstrating a greater effect in the groups of mice treated with the pharmaceutical composition of the peptide identified as SEQ ID NO: 1, where sucrose was added in concentrations of 20 and 30 mg / mL ( Figure 1 B) .
  • sucrose in concentrations in the range of 20-30 mg / mL is a determining factor for the induction of regulatory T cells, which control the magnitude of inflammation, as well as that this formulation applied intravenously, it has the capacity to control pathologies characterized by hyperinflammation.

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Abstract

Disclosed is a pharmaceutical composition comprising the APL peptide identified as SEQ ID NO. 1, as an inhibitor of a hyperinflammatory state characterised by an increase in cytokines or interleukins and inflammatory molecules, the concentration of which increases until reaching a state defined as "cytokine storm". The sucrose concentrations in the composition increase the biological activity of the peptide, facilitating its intravenous application. Said peptide in a pharmaceutical composition is useful for the production of a drug for treating hyperinflammation-related diseases, such as COVID-19, dengue, macrophage activation syndrome, respiratory sepsis and diseases associated with chimeric antigen receptor T-cell therapies, and acute respiratory distress syndrome. Also disclosed is a method for treating said diseases by administering a therapeutically effective amount of the composition.

Description

PÉPTIDO PARA EL TRATAMIENTO DEL SÍNDROME DE LA TORMENTA DE PEPTIDE FOR THE TREATMENT OF STORM SYNDROME
CITOCINAS CYTOKINES
Campo de la técnica La presente invención se relaciona con la rama de la biomedicina y la industria farmacéutica, en particular con una composición farmacéutica para el tratamiento y la prevención del síndrome de la tormenta de citocinas. Dicha composición comprende el péptido identificado como SEQ ID NO: 1 , y puede ser empleada en el tratamiento del síndrome de distrés respiratorio agudo, el síndrome de activación de macrófagos, la sepsis respiratoria, el dengue, la encefalopatía hepática y la pericarditis, entre otras enfermedades. Technical field The present invention relates to the branch of biomedicine and the pharmaceutical industry, in particular with a pharmaceutical composition for the treatment and prevention of cytokine storm syndrome. Said composition comprises the peptide identified as SEQ ID NO: 1, and can be used in the treatment of acute respiratory distress syndrome, macrophage activation syndrome, respiratory sepsis, dengue, hepatic encephalopathy and pericarditis, among others. diseases.
Estado de la técnica anterior Prior state of the art
Dentro de los individuos infectados con el virus SARS-CoV-2 que desarrollan la enfermedad infecciosa Covid-19, se ha descrito un subgrupo de pacientes en los que se genera un estado de hiperinflamación. En estos pacientes se describe una “tormenta de citocinas”, en particular de interleucinas (IL), caracterizada por un aumento de la IL-2, IL-17, IL-6, TNFa, entre otras. Este estado de hiperinflamación conduce a la muerte de los pacientes (Mehta, et al. (2020). The Lancet, 395: 1033- 1034). Por otro lado, en un estudio retrospectivo, en el que se incluyeron 150 casos confirmados con Covid-19 en Wuhan, China, se demostró un aumento significativo de la concentración de la ferritina en los pacientes que no sobrevivieron (1297,6 ng/ml), en comparación con los niveles de esta proteína en los pacientes que sobrevivieron a la enfermedad (614 ng/ml) (Rúan et al. (2020). Intensive Care Med; https://doi.Org/10.1007/s00134-020-05991 -x). Within the individuals infected with the SARS-CoV-2 virus who develop the infectious disease Covid-19, a subgroup of patients has been described in whom a state of hyperinflammation is generated. In these patients, a “cytokine storm” is described, in particular of interleukins (IL), characterized by an increase in IL-2, IL-17, IL-6, TNFa, among others. This state of hyperinflammation leads to the death of patients (Mehta, et al. (2020). The Lancet, 395: 1033-1034). On the other hand, in a retrospective study, which included 150 confirmed cases with Covid-19 in Wuhan, China, a significant increase in ferritin concentration was demonstrated in patients who did not survive (1297.6 ng / ml ), compared to the levels of this protein in patients who survived the disease (614 ng / ml) (Ruan et al. (2020). Intensive Care Med; https://doi.Org/10.1007/s00134-020 -05991 -x).
Se conoce que los pacientes en estado grave y crítico afectados por la Covid-19 desarrollan el síndrome de distrés respiratorio agudo. Este se caracteriza por la presencia de eventos inflamatorios en el alveolo pulmonar, que se expanden por la circulación sistémica del organismo. En el curso de este síndrome se produce una afectación endotelial pulmonar, con incremento de la permeabilidad vascular, y del tráfico de células y macromoléculas al espacio alveolar, donde inactivan el surfactante pulmonar, estableciendo las típicas membranas hialinas (Janz y Ware (2014). Clin Chest Med; 35: 685-96). La barrera epitelial también se altera, y se exacerba en esta una respuesta inflamatoria con secreción de IL proinflamatorias, como TNFa, IL-1 b, e IL-6, y de otros mediadores de la inflamación, con la sucesiva activación de neutrófilos, monocitos, macrófagos, y el tráfico de estas moléculas y células hacia los espacios alveolares, incrementando la afectación inicial. El desarrollo de esta hiperinflamación se presenta en otras enfermedades como la sepsis respiratoria, el síndrome de activación de macrófagos, y la sepsis en adultos y neonatos. It is known that patients in serious and critical condition affected by Covid-19 develop acute respiratory distress syndrome. This is characterized by the presence of inflammatory events in the pulmonary alveolus, which are expanded by the systemic circulation of the organism. In the course of this syndrome, pulmonary endothelial involvement occurs, with increased vascular permeability, and the traffic of cells and macromolecules to the alveolar space, where they inactivate the pulmonary surfactant, establishing the typical hyaline membranes (Janz and Ware (2014). Clin Chest Med; 35: 685-96). The epithelial barrier is also altered, and it exacerbates an inflammatory response with secretion of pro-inflammatory IL, such as TNFa, IL-1b, and IL-6, and other mediators of inflammation, with the successive activation of neutrophils, monocytes, macrophages, and the trafficking of these molecules and cells towards the alveolar spaces, increasing the initial involvement. The development of this hyperinflammation occurs in other diseases such as respiratory sepsis, macrophage activation syndrome, and sepsis in adults and neonates.
Hasta la fecha, no existe una terapia eficaz para los pacientes con la enfermedad Covid-19. Se están utilizando terapias aprobadas para el tratamiento de otras enfermedades, como la administración de anticuerpos monoclonales contra la IL-1 (Anakinra), la IL-6 (Tocilizumab), y los inhibidores de la proteína Janus quinasa. Sin embargo, estos fármacos no han sido eficaces, y además son inmunosupresores, por lo que el uso de estas terapias para tratar la Covid-19 puede conllevar a un empeoramiento del estado general del paciente, ya que se ha comprobado la persistencia viral durante el transcurso de la enfermedad, y aun después de la recuperación clínica del paciente. Tampoco existe una cura efectiva y segura para la sepsis respiratoria, el síndrome de activación de macrófagos, la sepsis neonatal, así como para el distrés respiratorio. To date, there is no effective therapy for patients with Covid-19 disease. Approved therapies are being used for the treatment of other diseases, such as the administration of monoclonal antibodies against IL-1 (Anakinra), IL-6 (Tocilizumab), and Janus protein kinase inhibitors. However, these drugs have not been effective, and they are also immunosuppressive, so the use of these therapies to treat Covid-19 can lead to a worsening of the general condition of the patient, since viral persistence has been proven during course of the disease, and even after the patient's clinical recovery. There is also no effective and safe cure for respiratory sepsis, macrophage activation syndrome, neonatal sepsis, as well as for respiratory distress.
Por tanto, es de gran interés la obtención de fármacos que sean eficaces para el tratamiento de la hiperinflamación y la “tormenta de citocinas” que caracteriza estas patologías. Therefore, it is of great interest to obtain drugs that are effective for the treatment of hyperinflammation and the "cytokine storm" that characterizes these pathologies.
Explicación de la invención Explanation of the invention
La presente invención resuelve el problema antes planteado, al proveer una composición farmacéutica para el tratamiento y la prevención del síndrome de la tormenta de citocinas que comprende una concentración de 1 ,8 - 3,6 mg/mL del péptido identificado como SEQ ID NO: 1 y una concentración de sacarosa entre 20- 30 mg/mL, lo que sorprendentemente, aumenta de forma sinérgica la actividad biológica de dicho péptido, además de resultar un factor determinante para la inducción de las células T reguladoras, las cuales controlan la magnitud de la inflamación. La composición de la invención es capaz de reducir la hiperinflamación que caracteriza el subgrupo de pacientes con la Covid-19 que transitan a un estadio grave, caracterizado por un distrés respiratorio, como sucede en la sepsis respiratoria, el síndrome de activación de macrófagos, y la sepsis neonatal y del adulto. La administración del péptido identificado como SEQ ID NO: 1 reduce el nivel de citocinas proinflamatorias en los pacientes con la enfermedad Covid-19, sin causar inmunosupresión ni efectos adversos serios o asociados a la terapia con el péptido. The present invention solves the problem raised above, by providing a pharmaceutical composition for the treatment and prevention of cytokine storm syndrome that comprises a concentration of 1.8-3.6 mg / mL of the peptide identified as SEQ ID NO: 1 and a sucrose concentration between 20-30 mg / mL, which surprisingly, synergistically increases the biological activity of said peptide, in addition to being a determining factor for the induction of regulatory T cells, which control the magnitude of the inflammation. The composition of the invention is capable of reducing the hyperinflammation that characterizes the subgroup of patients with Covid-19 who transition to a severe stage, characterized by respiratory distress, as occurs in respiratory sepsis, macrophage activation syndrome, and neonatal sepsis and adult. Administration of the peptide identified as SEQ ID NO: 1 reduces the level of pro-inflammatory cytokines in patients with Covid-19 disease, without causing immunosuppression or serious adverse effects or those associated with the peptide therapy.
El péptido identificado como SEQ ID NO: 1 se deriva de una región de la proteína HSP60, comprendida entre los aminoácidos del 83 al 109. La HSP60 es muy inmunogénica, tanto en individuos sanos como en pacientes con enfermedades autoinmunes. Sin embargo, el nivel de anticuerpos y células T que esta proteína puede inducir es mayor en los pacientes con enfermedades autoinmunes (de Jong etal { 2009). Arthritis Rheum. 7 (60): 1966-1976). Esta proteína tiene gran capacidad para estimular la reactividad proinflamatoria del sistema inmunitario innato. También se ha encontrado que la HSP60 induce la expresión de los genes de la IL-12 y la IL- 15, citocinas involucradas en la inducción de un fenotipo Th1. Estos hechos sugieren que la HSP60 actúa como una señal de peligro para el sistema inmunitario innato (Habich et al { 2005). J.lmmunol. 3 (174): 1298-1305). Por otra parte, péptidos derivados de la HSP60 también pueden constituir una señal de peligro (Quintana y Cohén (2011). Trends Immunol. 2 (32): 89-95). The peptide identified as SEQ ID NO: 1 is derived from a region of the HSP60 protein, comprised between amino acids 83 to 109. HSP60 is highly immunogenic, both in healthy individuals and in patients with autoimmune diseases. However, the level of antibodies and T cells that this protein can induce is higher in patients with autoimmune diseases (de Jong et al {2009). Arthritis Rheum. 7 (60): 1966-1976). This protein has a great capacity to stimulate the pro-inflammatory reactivity of the innate immune system. HSP60 has also been found to induce the expression of IL-12 and IL-15 genes, cytokines involved in the induction of a Th1 phenotype. These facts suggest that HSP60 acts as a danger signal for the innate immune system (Habich et al {2005). J.lmmunol. 3 (174): 1298-1305). On the other hand, peptides derived from HSP60 can also constitute a danger signal (Quintana and Cohén (2011). Trends Immunol. 2 (32): 89-95).
El péptido de SEQ ID NO: 1 contiene varios epitopos superpuestos de células T. El mismo incrementa la frecuencia de las células Treg con fenotipo CD4+CD25highFoxp3+ en ensayos ex vivo con células mononucleares de sangre periférica de pacientes con artritis reumatoide. Sin embargo, no ocurre así con células de donantes sanos (Barbera et al (2016). Cell Stress. Chaperones 4 (21): 735-744). The peptide of SEQ ID NO: 1 contains several overlapping T-cell epitopes. It increases the frequency of Treg cells with CD4 + CD25 high Foxp3 + phenotype in ex vivo assays with peripheral blood mononuclear cells from rheumatoid arthritis patients. However, this is not the case with cells from healthy donors (Barbera et al (2016). Cell Stress. Chaperones 4 (21): 735-744).
En las solicitudes internacionales de patente No. PCT/CU2005/000008 y PCT/CU2009/000009 se reivindica el péptido identificado como SEQ ID NO: 1 y su uso para el tratamiento de la artritis reumatoide, y de la enfermedad de Crohn, la colitis ulcerosa y la diabetes mellitus tipo I, respectivamente. Todas estas patologías son no trasmisibles. Además, la solicitud de patente WO2019/129315 describe una composición farmacéutica muy estable que contiene el péptido de SEQ ID NO: 1 , su administración causa la disminución de anticuerpos contra péptidos citrulinados y de otros parámetros muy asociados a la citrulinación de proteínas. International patent applications No. PCT / CU2005 / 000008 and PCT / CU2009 / 000009 claim the peptide identified as SEQ ID NO: 1 and its use for the treatment of rheumatoid arthritis, and Crohn's disease, colitis ulcerative and type I diabetes mellitus, respectively. All these pathologies are non-communicable. Furthermore, patent application WO2019 / 129315 describes a very stable pharmaceutical composition containing the peptide of SEQ ID NO: 1, its administration causes the reduction of antibodies against citrullinated peptides and of other parameters closely associated with the citrullination of proteins.
El péptido identificado como SEQ ID NO: 1 se obtiene por síntesis química. Mediante experimentos como los que se describen en la presente invención, se pueden evaluar aquellos mecanismos inmunológicos que induce el péptido que participan en el control de la hiperinflamación. La composición farmacéutica fue bien tolerada y con un alto perfil de seguridad, sin evidenciarse signos de inmunosupresión. No se identificaron eventos adversos graves en ningún paciente.The peptide identified as SEQ ID NO: 1 is obtained by chemical synthesis. Through experiments such as those described in the present invention, those immunological mechanisms induced by the peptide that they participate in the control of hyperinflammation. The pharmaceutical composition was well tolerated and with a high safety profile, with no evidence of immunosuppression. No serious adverse events were identified in any patient.
A los efectos de la invención se entiende por síndrome de la tormenta de citocinas al aumento de citocinas proinflamatorias, donde se alcanzan concentraciones que rebasan los niveles normales. Estas citocinas proinflamatorias son IL-6, TNFa, IL-1 , IL-17, entre otras. También se refiere al aumento de otras citocinas, como la IL-10, IL-8 e IL-5. Además, comprende el aumento de biomarcadores de inflamación, como la proteína C reactiva, el fibrinógeno, el Dímero-D, las transaminasas hepáticas, la lactato deshidrogenasa y la ferritina. El aumento de estas moléculas asociadas a la tormenta de citocinas ocurre en pacientes afectados de patologías inflamatorias como: distrés respiratorio agudo, el síndrome de activación de macrófagos, la sepsis respiratoria, el dengue, la encefalopatía hepática y la pericarditis. Desde el punto de vista clínico, puede evidenciarse en los pacientes fiebre sostenida, decaimiento, fatiga y náuseas. For the purposes of the invention, cytokine storm syndrome is understood to be the increase in pro-inflammatory cytokines, where concentrations that exceed normal levels are reached. These pro-inflammatory cytokines are IL-6, TNFa, IL-1, IL-17, among others. It also refers to the increase in other cytokines, such as IL-10, IL-8, and IL-5. In addition, it includes the increase in biomarkers of inflammation, such as C-reactive protein, fibrinogen, D-dimer, liver transaminases, lactate dehydrogenase and ferritin. The increase in these molecules associated with the cytokine storm occurs in patients affected by inflammatory pathologies such as: acute respiratory distress, macrophage activation syndrome, respiratory sepsis, dengue, hepatic encephalopathy and pericarditis. From the clinical point of view, sustained fever, weakness, fatigue and nausea can be evidenced in patients.
En otro aspecto, la invención revela el uso de una composición farmacéutica que comprende el péptido identificado como SEQ ID NO: 1 en la fabricación de un medicamento para el tratamiento y la prevención del síndrome de la tormenta de citocinas. Sorprendentemente, el tratamiento con una composición que comprende dicho péptido permite la retirada de la ventilación mecánica de los pacientes con la Covid-19, a partir de la mejoría de la neumonía intersticial, la disminución de los reactantes de la fase aguda de la inflamación entre las 24 y 48 horas, y la reducción de los biomarcadores de la inflamación. La disminución rápida de la proteína C reactiva, en tan solo 24 horas, es un hallazgo inesperado. Anteriormente, en pacientes con artritis reumatoide que se trataron con este péptido, no se logró demostrar una disminución significativa de la proteína C reactiva (Prada, et al (2018). J Clin Triáis 1 (8): 1-11). In another aspect, the invention discloses the use of a pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1 in the manufacture of a medicament for the treatment and prevention of cytokine storm syndrome. Surprisingly, treatment with a composition comprising said peptide allows the withdrawal of mechanical ventilation in patients with Covid-19, from the improvement of interstitial pneumonia, the reduction of reactants of the acute phase of inflammation among 24 and 48 hours, and the reduction of biomarkers of inflammation. The rapid decrease in C-reactive protein, in just 24 hours, is an unexpected finding. Previously, in patients with rheumatoid arthritis who were treated with this peptide, it was not possible to demonstrate a significant decrease in C-reactive protein (Prada, et al (2018). J Clin Triáis 1 (8): 1-11).
En la presente invención se demuestra la capacidad del péptido identificado como SEQ ID NO: 1, administrado por vía intravenosa, de reducir el distrés respiratorio, la hiperinflamación, los reactantes de la fase aguda de la inflamación, y el nivel de citocinas proinflamatorias en los pacientes con la Covid-19. Sorprendentemente, estos resultados se reprodujeron en pacientes con sepsis respiratoria que presentaron el síndrome de distrés respiratorio. En una realización particular, la composición farmacéutica que comprende el péptido identificado como SEQ ID NO: 1 se utiliza en la fabricación de un medicamento para el tratamiento y la prevención del síndrome de la tormenta de citocinas que se manifiesta como síndrome de distrés respiratorio agudo, síndrome de activación de macrófagos, sepsis respiratoria en adultos o neonatos, síndrome de respuesta inflamatoria sistémica, tromboembolismo venoso o arterial, elevación aguda de los niveles de ferritina, pericarditis severa, elevación aguda de las transaminasas hepáticas que causan encefalopatía hepática o enfermedad asociada a las terapias con células T que poseen receptor de antígeno quimérico. Dentro de las patologías que se tratan con la composición farmacéutica que comprende el péptido identificado como SEQ ID NO: 1 está el síndrome de la tormenta de citocinas que subyace en el síndrome de respuesta inflamatoria sistémica, que se produce en los pacientes tras las quemaduras o traumatismos severos o en enfermedades inflamatorias agudas. En una materialización de la invención, el medicamento se administra por vía intravenosa. The present invention demonstrates the ability of the peptide identified as SEQ ID NO: 1, administered intravenously, to reduce respiratory distress, hyperinflammation, the reactants of the acute phase of inflammation, and the level of pro-inflammatory cytokines in the patients with Covid-19. Surprisingly, these results were reproduced in patients with respiratory sepsis who presented with the respiratory distress syndrome. In a particular embodiment, the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1 is used in the manufacture of a drug for the treatment and prevention of cytokine storm syndrome that manifests as acute respiratory distress syndrome, macrophage activation syndrome, respiratory sepsis in adults or neonates, systemic inflammatory response syndrome, venous or arterial thromboembolism, acute elevation of ferritin levels, severe pericarditis, acute elevation of liver transaminases causing hepatic encephalopathy or disease associated with therapies with T cells that possess chimeric antigen receptor. Among the pathologies that are treated with the pharmaceutical composition that comprises the peptide identified as SEQ ID NO: 1 is the cytokine storm syndrome that underlies the systemic inflammatory response syndrome, which occurs in patients after burns or severe trauma or in acute inflammatory diseases. In one embodiment of the invention, the drug is administered intravenously.
En una realización de la invención, el medicamento se emplea para el tratamiento y la prevención del síndrome de la tormenta de citocinas causado por una enfermedad infecciosa. En una realización particular, dicha enfermedad infecciosa es la Covid-19 o el dengue. Es objeto de la presente invención un método para el tratamiento o prevención del síndrome de la tormenta de citocinas que comprende la administración de una cantidad farmacéuticamente efectiva de una composición farmacéutica que comprende el péptido identificado como SEQ ID NO: 1 , a un individuo que lo necesita. En una realización particular, el síndrome de la tormenta de citocinas se manifiesta como síndrome de distrés respiratorio agudo, síndrome de activación de macrófagos, sepsis respiratoria en adultos o neonatos, síndrome de respuesta inflamatoria sistémica, tromboembolismo venoso o arterial, elevación aguda de los niveles de ferritina, pericarditis severa, elevación aguda de las transaminasas hepáticas que causan encefalopatía hepática o enfermedad asociada a las terapias con células T que poseen receptor de antígeno quimérico. En una realización, la tormenta de citocinas que se trata o se previene con el método de la invención es causada por una enfermedad infecciosa. En una realización preferida el método de la invención es útil para el tratamiento de la enfermedad infecciosa Covid-19 o dengue. En una materialización de la invención, en dicho método la composición se administra por vía intravenosa. In one embodiment of the invention, the medicament is used for the treatment and prevention of cytokine storm syndrome caused by an infectious disease. In a particular embodiment, said infectious disease is Covid-19 or dengue. The object of the present invention is a method for the treatment or prevention of cytokine storm syndrome that comprises the administration of a pharmaceutically effective amount of a pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1, to an individual who does so. needs to. In a particular embodiment, the cytokine storm syndrome manifests as acute respiratory distress syndrome, macrophage activation syndrome, respiratory sepsis in adults or neonates, systemic inflammatory response syndrome, venous or arterial thromboembolism, acute elevation of levels. of ferritin, severe pericarditis, acute elevation of the hepatic transaminases that cause hepatic encephalopathy or disease associated to the therapies with T cells that possess chimeric antigen receptor. In one embodiment, the cytokine storm that is treated or prevented by the method of the invention is caused by an infectious disease. In a preferred embodiment the method of the invention is useful for the treatment of the infectious disease Covid-19 or dengue. In one embodiment of the invention, in said method the composition is administered intravenously.
La administración de la composición farmacéutica que comprende el péptido identificado como SEQ ID NO: 1 a pacientes que lo necesitan disminuye los eventos asociados con un distrés respiratorio. Este distrés provoca que los pacientes tengan necesidad de: The administration of the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1 to patients in need thereof reduces the events associated with respiratory distress. This distress causes patients to need:
• Oxigenoterapia para mantener saturación de oxígeno (S02) por encima del 93% • Oxygen therapy to maintain oxygen saturation (S0 2 ) above 93%
• Saturación de oxígeno mayor a 3 puntos porcentuales o disminución en la presión parcial de oxígeno arterial (Pa02) mayor al 10%, con fracción de oxígeno inspirada (Fi02) estable en las últimas 24 horas • Oxygen saturation greater than 3 percentage points or decrease in arterial partial pressure of oxygen (Pa0 2 ) greater than 10%, with a stable fraction of inspired oxygen (Fi0 2 ) in the last 24 hours
• Aumento de Fi02 con el fin de mantener un S02 estable o nueva necesidad de ventilación mecánica en las últimas 24 horas • Increase in Fi0 2 in order to maintain a stable S0 2 or new need for mechanical ventilation in the last 24 hours
• Aumento del número y/o extensión de las áreas pulmonares de consolidación Además, la administración de la composición de la invención es útil para el tratamiento de pacientes afectados con la enfermedad infecciosa Covid-19, cuando dichos pacientes tienen necesidad de oxigenoterapia no menor de 6 L/min más una de las siguientes condiciones: • Increase in the number and / or extension of the lung areas of consolidation In addition, the administration of the composition of the invention is useful for the treatment of patients affected with the infectious disease Covid-19, when said patients have a need for oxygen therapy of no less than 6 L / min plus one of the following conditions:
Sibilancia o habla entrecortada Wheezing or choppy speech
Frecuencia respiratoria mayor de 22 respiraciones/min con oxigenoterapia a L/min Respiratory rate greater than 22 breaths / min with oxygen therapy at L / min
Pa02: Menor de 65 mm Hg Empeoramiento de la imagen radiológica Fiebre mayor o igual a 39QC Pa0 2 : Less than 65 mm Hg Worsening of the radiological image Fever greater than or equal to 39 Q C
Reducción de los valores iniciales de hemoglobina, plaquetas ó leucocitos hemoglobina <9,2 gm/dl, leucocitos <5000/mm3, plaquetas<110,000/mmReduction in baseline hemoglobin, platelets or leukocytes hemoglobin <9.2 gm / dl, leukocytes <5000 / mm 3 , platelets <110,000 / mm
Disminución de la eritrosedimentación en discordancia con la proteína C eactiva que aumenta o no se modifica Decreased erythrocyte sedimentation in disagreement with C eactive protein that is increased or unchanged
Aumento del valor inicial de los triglicéridos por encima de 3 mmol/L Aumento del valor inicial de la ferritina a partir de 500 ng/ml o valor absoluto de ferritina mayor o igual a 2000 ng/ml Increase in the initial value of triglycerides above 3 mmol / L Increase in the initial value of ferritin from 500 ng / ml or absolute value of ferritin greater than or equal to 2000 ng / ml
Transaminasa aspartato-aminotransferasa mayor o igual a 30 Ul/L Aumento del dímero D Fibrinógeno menor de 2,5 g/L • Aparición de manifestaciones neurológicas Aspartate-aminotransferase transaminase greater than or equal to 30 IU / L Increase in D-dimer Fibrinogen less than 2.5 g / L • Appearance of neurological manifestations
La composición de la invención también es útil para el tratamiento de los pacientes con la Covid-19 que entran en un estado grave sin necesidad de oxigenoterapia, pero que presentan un aumento del valor inicial de la ferritina a partir de 500 ng/ml o valor absoluto de ferritina mayor o igual a 2000 ng/ml, con signos radiológicos progresivos de neumonía intersticial multifocal. The composition of the invention is also useful for the treatment of patients with Covid-19 who enter a serious state without the need for oxygen therapy, but who present an increase in the initial value of ferritin from 500 ng / ml or value absolute ferritin greater than or equal to 2000 ng / ml, with progressive radiological signs of multifocal interstitial pneumonia.
También es útil para el tratamiento de enfermedades caracterizadas por un estado de hiperinflamación aguda como el que desarrollan la sepsis respiratoria en el adulto o en los neonatos; y para las enfermedades que desarrollan el síndrome de distrés respiratorio, así como para dicho síndrome provocado por causas mecánicas y físicas. It is also useful for the treatment of diseases characterized by a state of acute hyperinflammation such as that which develops respiratory sepsis in adults or neonates; and for diseases that develop respiratory distress syndrome, as well as for said syndrome caused by mechanical and physical causes.
Breve descripción de las figuras Brief description of the figures
Figura 1A. Frecuencia de células T cuantificados por citometría de flujo que expresan el fenotipo CD4+ Foxp3+ , aisladas del bazo de ratones BALB/c, después ser inoculados con la composición farmacéutica del péptido identificado como SEQ ID NO: 1 , donde la sacarosa se adicionó a una concentración de 10 mg/mL. (10 ), de 30 mg/mL (30) y 80 mg/mL (80) . Para el análisis de los datos se emplearon las pruebas estadísticas Kruskal-Wallis y Dunn. Letras diferentes indican diferencias estadísticamente significativas (P<0.05). Figure 1A. Frequency of T cells quantified by flow cytometry expressing the CD4 + Foxp3 + phenotype, isolated from the spleen of BALB / c mice, after being inoculated with the pharmaceutical composition of the peptide identified as SEQ ID NO: 1, where sucrose was added at a concentration 10 mg / mL. (10), 30 mg / mL (30) and 80 mg / mL (80). The Kruskal-Wallis and Dunn statistical tests were used for data analysis. Different letters indicate statistically significant differences (P <0.05).
Figura 1B. Frecuencia de células T cuantificados por citometría de flujo que expresan el fenotipo CD4+ Foxp3+ , aisladas del bazo de ratones BALB/c, después ser inoculados con la composición farmacéutica del péptido identificado como SEQ ID NO: 1 , donde la sacarosa se adicionó a una concentración de 20 mg/mL. (20 ), 30 mg/mL (30) y 40 mg/mL (40) . Para el análisis de los datos se emplearon las pruebas estadísticas Kruskal-Wallis y Dunn. Letras diferentes indican diferencias estadísticamente significativas (P<0.05). Figure 1B. Frequency of T cells quantified by flow cytometry expressing the CD4 + Foxp3 + phenotype, isolated from the spleen of BALB / c mice, after being inoculated with the pharmaceutical composition of the peptide identified as SEQ ID NO: 1, where sucrose was added at a concentration of 20 mg / mL. (20), 30 mg / mL (30) and 40 mg / mL (40). The Kruskal-Wallis and Dunn statistical tests were used for data analysis. Different letters indicate statistically significant differences (P <0.05).
Exposición detallada de modos de realización / Ejemplos de realización Ejemplo 1. Tratamiento de pacientes con la enfermedad Covid-19 con la composición farmacéutica que comprende el péptido identificado como SEQ ID NO: 1. Detailed discussion of embodiments / Examples of embodiments Example 1. Treatment of patients with Covid-19 disease with the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1.
Se ejemplifica con diez pacientes ingresados en la Unidad de Cuidados Intensivos del Hospital Militar Luis Díaz Soto, afectados con la enfermedad infecciosa Covid- 19. Estos pacientes recibían atención médica según el protocolo establecido por el Ministerio de Salud Pública de Cuba para el tratamiento de esta enfermedad. Los pacientes desarrollaron el síndrome de distrés respiratorio agudo, y el mismo día que lo presentaron fueron sometidos a ventilación artificial mecánica/ventilación con liberación de presión en la vía aérea (VAM APRV). Después de recibir el consentimiento informado del representante legal del paciente, según lo establecido por la autoridad regulatoria cubana y el Comité de Ética y Revisión de dicho hospital, los pacientes recibieron la composición farmacéutica que comprende el péptido identificado como SEQ ID NO: 1. Dicho péptido se obtuvo por síntesis química, por un método bien conocido en este campo de la técnica La composición farmacéutica fue administrada por vía intravenosa, una vez cada 12 horas. En ocho de los diez pacientes, se administró 1 mg del péptido por paciente en cada aplicación. Los pacientes con el código 5 y 6 recibieron 2 mg cada 12 horas, ya que tenían cáncer entre las comorbilidades. Las características de los pacientes, así como los días de VAM APRV y de administración del péptido se reflejan en la Tabla 1. It is exemplified with ten patients admitted to the Intensive Care Unit of the Luis Díaz Soto Military Hospital, affected with the infectious disease Covid-19. These patients received medical attention according to the protocol established by the Ministry of Public Health of Cuba for the treatment of this disease. The patients developed acute respiratory distress syndrome, and on the same day they presented it, they underwent mechanical ventilation / airway pressure release ventilation (VAM APRV). After receiving the informed consent of the patient's legal representative, as established by the Cuban regulatory authority and the Ethics and Review Committee of said hospital, the patients received the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1. Said The peptide was obtained by chemical synthesis, by a method well known in this field of the art. The pharmaceutical composition was administered intravenously, once every 12 hours. In eight of the ten patients, 1 mg of the peptide was administered per patient in each application. Patients with code 5 and 6 received 2 mg every 12 hours, as they had cancer among the comorbidities. The characteristics of the patients, as well as the days of VAM APRV and administration of the peptide are reflected in Table 1.
Tabla 1. Características de los pacientes tratados con la composición farmacéutica que comprende el péptido identificado como SEQ ID NO: 1.
Figure imgf000010_0001
Figure imgf000011_0001
Table 1. Characteristics of the patients treated with the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1.
Figure imgf000010_0001
Figure imgf000011_0001
HTA: hipertensión arterial. VAM APRV: Ventilación artificial mecánica/ventilación con liberación de presión en la vía aérea. Como se aprecia en la tabla, los diez pacientes con la enfermedad Covid-19 que se trataron con el péptido tenían factores de riesgo que predisponen a un estado de gravedad o crítico de la misma. Un solo paciente no presentaba comorbilidades, pero su avanzada edad (80 años) constituye un factor de riesgo. En particular, la paciente identificada con el no. 5 presentó una trombosis venosa profunda. Sin embargo, la evolución clínica de los diez, una vez que se les administró el péptido, fue favorable. En el momento en que se procede a la extubación se considera que finaliza el síndrome de distrés respiratorio agudo. Estos resultados demuestran que el tratamiento con el péptido fue efectivo para el síndrome de distrés respiratorio agudo que desarrollan estos pacientes. La desaparición de dicho síndrome en los pacientes coincidió con la mejoría clínica y radiológica de los mismos. La paciente que presentó la trombosis venosa profunda evolucionó de forma favorable también. Estos pacientes no presentaron ningún síntoma de inmunosupresión. Antes de la terapia presentaban linfopenia, signo característico de los pacientes con Covid-19. Producto del tratamiento con el péptido los niveles de linfocitos circulantes se recuperaron paulatinamente hasta la normalidad. HT: arterial hypertension. VAM APRV: Mechanical Artificial Ventilation / Airway Pressure Release Ventilation. As can be seen in the table, the ten patients with Covid-19 disease who were treated with the peptide had risk factors that predispose to a serious or critical state of the same. Only one patient had no comorbidities, but his advanced age (80 years) was a risk factor. In particular, the patient identified with no. 5 presented a deep vein thrombosis. However, the clinical evolution of the ten, once they were administered the peptide, was favorable. At the time of extubation, the acute respiratory distress syndrome is considered to have ended. These results demonstrate that the peptide treatment was effective for the acute respiratory distress syndrome that these patients develop. The disappearance of this syndrome in the patients coincided with their clinical and radiological improvement. The patient who presented with deep vein thrombosis also evolved favorably. These patients did not present any symptoms of immunosuppression. Before therapy, they presented lymphopenia, a characteristic sign of patients with Covid-19. As a result of the peptide treatment, the levels of circulating lymphocytes gradually recovered to normality.
La literatura especializada refiere que el 85%-90% de los pacientes con síndrome de distrés respiratorio agudo no sobreviven. En Cuba, dentro de los pacientes confirmados con SAR-COV-2 que transitaron hacia un estadio crítico o grave, hubo una tasa de letalidad de 68,6% en los individuos que no fueron tratados con la composición farmacéutica que comprende el péptido identificado como SEQ ID NO: 1. En contraste, la tasa de letalidad fue de 13,0% en los pacientes críticos o graves, confirmados con SAR-COV-2, que se trataron con la composición farmacéutica que comprende el péptido identificado como SEQ ID NO: 1. Ejemplo 2. Administración de la composición farmacéutica que comprende el péptido identificado como SEQ ID NO: 1 para prevenir el establecimiento del síndrome de distrés respiratorio. The specialized literature reports that 85% -90% of patients with acute respiratory distress syndrome do not survive. In Cuba, among patients confirmed with SAR-COV-2 who transited to a critical or severe stage, there was a fatality rate of 68.6% in individuals who were not treated with the pharmaceutical composition that comprises the peptide identified as SEQ ID NO: 1. In contrast, the fatality rate was 13.0% in critical or seriously ill patients, confirmed with SAR-COV-2, who were treated with the pharmaceutical composition comprising the peptide identified as SEQ ID NO : 1. Example 2. Administration of the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1 to prevent the establishment of respiratory distress syndrome.
Los pacientes en estado grave y críticos afectados de la Covid-19 desarrollan el síndrome de distrés respiratorio, que puede ocasionar la muerte. De forma sorprendente, la administración de la composición farmacéutica que comprende el péptido identificado como SEQ ID NO: 1 previno el desarrollo de un síndrome de distrés respiratorio agudo. El péptido se administró en una etapa temprana, luego de los primeros signos de alarma (indicadores radiológicos progresivos de neumonía intersticial multifocal, ferritina elevada (2 veces por encima del valor normal), oximetría de pulso inferior a 94 de saturación de oxígeno). La administración de dicho péptido previno, por tanto, la necesidad de ventilación artificial. De esta forma, se evitó la probable evolución a una sepsis nosocomial. Critically ill and critically ill patients from Covid-19 develop respiratory distress syndrome, which can lead to death. Surprisingly, the administration of the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1 prevented the development of an acute respiratory distress syndrome. The peptide was administered at an early stage, after the first warning signs (progressive radiological indicators of multifocal interstitial pneumonia, elevated ferritin (2 times above normal value), pulse oximetry below 94 oxygen saturation). The administration of said peptide therefore prevented the need for artificial ventilation. In this way, the probable evolution to nosocomial sepsis was avoided.
Se ejemplifica con un paciente de 42 años, del sexo femenino, afectado con la enfermedad Covid-19, ingresado en la sala de terapia intermedia del Hospital del Instituto de Medicina Tropical “Pedro Kouri”. Este paciente recibía atención médica, según el protocolo establecido por el Ministerio de Salud Pública de Cuba para el tratamiento de esta enfermedad, pero presentó un cuadro de disnea repentino, con fiebre de 39 QC y una concentración de ferritina de 700 ng/mL. Con el consentimiento informado, se le realizó la administración de la composición farmacéutica que contiene el péptido identificado como SEQ ID NO: 1 , por vía intravenosa, en una dosis de 1 mg, cada 12 horas. Al cabo de las 2 horas, el cuadro de disnea desapareció y la temperatura corporal disminuyó. El paciente no desarrolló el síndrome de distrés respiratorio agudo, y en el transcurso de 72 horas la concentración de ferritina disminuyó por debajo de 500 ng/mL. It is exemplified with a 42-year-old female patient, affected with Covid-19 disease, admitted to the intermediate therapy room of the Hospital of the “Pedro Kouri” Institute of Tropical Medicine. This patient received medical attention, according to the protocol established by the Cuban Ministry of Public Health for the treatment of this disease, but he presented sudden dyspnea, with a fever of 39 Q C and a ferritin concentration of 700 ng / mL. With informed consent, the pharmaceutical composition containing the peptide identified as SEQ ID NO: 1 was administered intravenously, in a dose of 1 mg, every 12 hours. After 2 hours, the dyspnea condition disappeared and the body temperature decreased. The patient did not develop acute respiratory distress syndrome, and within 72 hours the ferritin concentration dropped below 500 ng / mL.
Además, se trataron otros tres pacientes en estado grave, cuyas edades y comorbilidades se presentan en la Tabla 2. Estos pacientes estaban ingresados en la sala de terapia intermedia del Hospital Militar Central Luis Díaz Soto. In addition, three other patients in serious condition were treated, whose ages and comorbidities are presented in Table 2. These patients were admitted to the intermediate therapy room of the Luis Díaz Soto Central Military Hospital.
Tabla 2. Edad y comorbilidades de los pacientes graves tratados con la composición farmacéutica que comprende el péptido identificado como SEQ ID NO: 1.
Figure imgf000013_0001
Table 2. Age and comorbidities of critically ill patients treated with the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1.
Figure imgf000013_0001
HTA: hipertensión arterial HT: arterial hypertension
Estos pacientes también presentaron los primeros signos de alarma, como: fiebre sostenida por encima de 38 QC, disnea con necesidad oxigenoterapia por máscara o tenedor nasal, para mantener una saturación de oxígeno por encima de 93. Presentaron, además, polipnea mayor de 25 ventilaciones por minuto y necesitaron oxigenoterapia por máscara o tenedor nasal, para mantener una saturación de oxígeno por encima de 93. These patients also had the early warning signs such as fever sustained above 38 Q C, dyspnea need for oxygen mask or nasal fork, to maintain oxygen saturation above 93. They presented also greater polipnea 25 ventilations per minute and required oxygen therapy by mask or nasal fork, to maintain an oxygen saturation above 93.
La administración de la composición farmacéutica que contiene el péptido identificado como SEQ ID NO: 1 , se realizó por vía intravenosa, en una dosis de 1 mg, cada 24 horas. Al cabo de las 2 horas de tratamiento, el cuadro de disnea desapareció, y la temperatura corporal disminuyó. Los pacientes no desarrollaron el síndrome de distrés respiratorio agudo, y en el transcurso de 72 horas la concentración de ferritina disminuyó por debajo de 200 ng/mL. Estos resultados demuestran que, el tratamiento de los pacientes con la enfermedad Covid-19 con la composición farmacéutica que contiene el péptido identificado como SEQ ID NO: 1 , antes de que desarrollen el síndrome de distrés respiratorio agudo, evita el uso de la ventilación mecánica para estos pacientes. The administration of the pharmaceutical composition containing the peptide identified as SEQ ID NO: 1, was carried out intravenously, in a dose of 1 mg, every 24 hours. After 2 hours of treatment, the dyspnea condition disappeared, and the body temperature decreased. The patients did not develop acute respiratory distress syndrome, and within 72 hours the ferritin concentration dropped below 200 ng / mL. These results demonstrate that the treatment of patients with Covid-19 disease with the pharmaceutical composition containing the peptide identified as SEQ ID NO: 1, before they develop acute respiratory distress syndrome, avoids the use of mechanical ventilation. for these patients.
Ejemplo 3. Disminución de la proteína C reactiva en los pacientes con la enfermedad Covid-19 tratados con la composición farmacéutica que comprende el péptido identificado como SEQ ID NO: 1. Example 3. Decrease in C-reactive protein in patients with Covid-19 disease treated with the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1.
La proteína C reactiva constituye uno de los marcadores más utilizados para los procesos inflamatorios, es considerada uno de los reactantes de la fase aguda de la inflamación. La composición farmacéutica que comprende el péptido identificado como SEQ ID NO: 1 se administró por vía intravenosa. Ocho de los diez pacientes afectados con la Covid-19, cuyo tratamiento se describe en el Ejemplo 1 , recibieron 1 mg del péptido cada 12 horas. Los pacientes con el código 5 y 6 que tenían entre las comorbilidades cáncer recibieron 2 mg del péptido cada 12 horas. Inesperadamente, el tratamiento con el péptido de SEQ ID NO: 1 disminuyó los niveles de proteína C reactiva, de forma marcada, en los pacientes tratados. Estos resultados se muestran en la Tabla 3. C-reactive protein is one of the most used markers for inflammatory processes, it is considered one of the reactants of the acute phase of inflammation. The pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1 was administered intravenously. Eight of the ten patients affected with Covid-19, whose treatment is described in Example 1, received 1 mg of the peptide every 12 hours. Patients with code 5 and 6 who had cancer between comorbidities received 2 mg of the peptide every 12 hours. Unexpectedly, treatment with the peptide of SEQ ID NO: 1 decreased the levels of C-reactive protein, markedly, in the treated patients. These results are shown in Table 3.
Tabla 3. Concentración de proteína C reactiva (mg/mL) en los pacientes tratados con el péptido identificado como SEQ ID NO: 1 .
Figure imgf000014_0001
Table 3. C-reactive protein concentration (mg / mL) in patients treated with the peptide identified as SEQ ID NO: 1.
Figure imgf000014_0001
No se rea izó la determinación. The determination was not made.
La disminución de proteína C reactiva durante el tratamiento de estos pacientes es un indicativo directo de que el péptido de SEQ ID NO: 1 disminuye la inflamación en los mismos, y está en correspondencia con la desaparición del síndrome de distrés respiratorio agudo, así como con la mejoría clínica y radiológica de los pacientes.The decrease in C-reactive protein during the treatment of these patients is a direct indication that the peptide of SEQ ID NO: 1 reduces inflammation in them, and is in correspondence with the disappearance of the acute respiratory distress syndrome, as well as with the clinical and radiological improvement of the patients.
Los resultados obtenidos en este estudio son sorprendentes. Durante la realización de un ensayo clínico, en pacientes con artritis reumatoide tratados con el mismo péptido, no se encontró disminución de dicho reactante de la fase aguda de la inflamación. The results obtained in this study are surprising. During a clinical trial, in patients with rheumatoid arthritis treated with the same peptide, no decrease in this reactant was found in the acute phase of inflammation.
Ejemplo 4. Disminución de citocinas proinflamatorias en pacientes con la enfermedad Covid-19 tratados con la composición farmacéutica que comprende el péptido identificado como SEQ ID NO: 1. Example 4. Decrease in pro-inflammatory cytokines in patients with Covid-19 disease treated with the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1.
En este estudio se determinaron los niveles de las citocinas proinflamatorias IL-6, IL- 17, IL-1 , IFNy y TNFa en pacientes con la enfermedad Covid-19 tratados con la composición que comprende el péptido identificado como SEQ ID NO: 1. Los pacientes se encontraban en estado crítico. El tratamiento de estos pacientes se enunció en el Ejemplo 1 . Dichas citocinas se cuantificaron en los sueros de los pacientes identificados con los números 1 , 2, 3, 4, 7 y 8, que aparecen en la Tabla 1 , Ejemplo 1. Para la determinación de las citocinas en las muestras de suero se inactivó el virus, bajo estrictas medidas de bioseguridad. La cuantificación se realizó justo antes de aplicar la primera dosis del péptido, y a las 48, 72 y 96 horas después de iniciado el tratamiento con el mismo. In this study, the levels of the pro-inflammatory cytokines IL-6, IL-17, IL-1, IFNy and TNFa were determined in patients with Covid-19 disease treated with the composition comprising the peptide identified as SEQ ID NO: 1. The patients were in critical condition. The treatment of these patients was set forth in Example 1. These cytokines were quantified in the sera of the patients identified with the numbers 1, 2, 3, 4, 7 and 8, which appear in Table 1, Example 1. For the determination of cytokines in serum samples, the virus, under strict biosecurity measures. The quantification was carried out just before applying the first dose of the peptide, and at 48, 72 and 96 hours after starting the treatment with it.
Todas las determinaciones de citocinas se realizaron mediante un inmunoensayo tipo ELISA (del inglés enzyme-linked immunosorbent assay) específico para cada una, siguiendo las recomendaciones del fabricante (Quantikine® R&D Systems, EEUU). Brevemente, se añadieron 50 pL del diluente especificado para cada citocina en los pocilios, y a continuación 200 pL de la curva patrón, la muestra o el control. Se incubó la placa durante 2 h a temperatura ambiente, y después se desechó el contenido de cada pocilio. Luego se realizaron tres lavados con la solución de lavado 1X, se añadieron 200 pL del anticuerpo conjugado a cada pocilio, y se incubó durante 1 h a temperatura ambiente. Se repitió el lavado y se añadieron 200 pL de la solución sustrato a cada pocilio. Se incubó la placa durante 20 min a temperatura ambiente protegida de la luz, se añadieron 50 pL de la solución de parada a cada pocilio, y se determinó la densidad óptica a 450 nm en un lector de microplacas (Thermo Scientific Multiskan Go, Finlandia). Se realizó una segunda lectura a 570 nm, y se sustrajeron los valores obtenidos a la lectura de 450 nm, para corregir las imperfecciones ópticas en la placa. La determinación de la concentración de cada citocina se realizó por triplicado. Los resultados obtenidos para IL-6 se muestran en la Tabla 4. All cytokine determinations were carried out using an ELISA (enzyme-linked immunosorbent assay) specific for each one, following the manufacturer's recommendations (Quantikine® R&D Systems, USA). Briefly, 50 pL of the specified diluent for each cytokine was added to the wells, followed by 200 pL of the standard curve, sample or control. The plate was incubated for 2 h at room temperature, and then the contents of each well were discarded. Then three washes were performed with the 1X wash solution, 200 pL of the conjugated antibody was added to each well, and incubated for 1 hr at room temperature. The wash was repeated and 200 pL of the substrate solution was added to each well. The plate was incubated for 20 min at room temperature protected from light, 50 pL of the stop solution was added to each well, and the optical density was determined at 450 nm in a microplate reader (Thermo Scientific Multiskan Go, Finland) . A second reading was made at 570 nm, and the values obtained were subtracted from the 450 nm reading, to correct the optical imperfections on the plate. The determination of the concentration of each cytokine was carried out in triplicate. The results obtained for IL-6 are shown in Table 4.
Tabla 4. Niveles de la citocina IL-6 (pg/mL) encontrados en los pacientes tratados con el péptido.
Figure imgf000015_0001
Como se aprecia en la tabla, hubo una disminución de los niveles de la citocina IL-6, a partir de que los pacientes en estado crítico recibieron el péptido de SEQ ID NO: 1. Esta es una citocina que aumenta marcadamente en los procesos inflamatorios. Se ha descrito que los pacientes con la enfermedad Covid-19 que transitan hacia un estadio grave tienen elevada esta citocina, en contraste con los pacientes que presentan síntomas leves de la enfermedad. De igual forma, se encontró en estos pacientes una disminución de IL-17, IL-1 , IFNy y TNFa. Estos resultados confirman que el tratamiento con el péptido disminuye la hiperinflamación, y la tormenta de citocinas, que desarrollan los pacientes con la enfermedad Covid-19 que transitan a un estadio grave. Table 4. IL-6 cytokine levels (pg / mL) found in patients treated with the peptide.
Figure imgf000015_0001
As can be seen in the table, there was a decrease in the levels of the cytokine IL-6, since the critically ill patients received the peptide of SEQ ID NO: 1. This is a cytokine that increases markedly in inflammatory processes . It has been described that patients with Covid-19 disease who transition to a severe stage have elevated this cytokine, in contrast to patients who present mild symptoms of the disease. Similarly, a decrease in IL-17, IL-1, IFNy and TNFa was found in these patients. These results confirm that treatment with the peptide reduces hyperinflammation, and cytokine storm, developed by patients with Covid-19 disease who are transitioning to a severe stage.
Ejemplo 5. Tratamiento de paciente con la enfermedad Covid-19 que presentó altas concentraciones de ferritina sin distrés respiratorio con la composición farmacéutica que comprende el péptido identificado como SEQ ID NO: 1. Example 5. Treatment of a patient with Covid-19 disease who presented high concentrations of ferritin without respiratory distress with the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1.
El paciente tratado tenía 66 años, era del sexo femenino, con antecedentes de diabetes mellitus tipo 2, obesidad exógena, hepatopatía grasa grado 4. Se ingresó, por diagnóstico de COVID-19, presentó un cuadro digestivo dado por dispepsias, nauseas, diarreas ocasionales, por lo que se le indicó procinéticos y anti-eméticos. Además, presentó altas concentraciones de ferritina, por encima de 1500 ng/mL, y presentó elevación de la velocidad de sedimentación globular con caída de la hemoglobina. Por este cuadro clínico los especialistas le indicaron tratamiento con la composición farmacéutica que comprende el péptido identificado como SEQ ID NO: 1 , a una dosis de 1 mg, cada 12 horas. The patient treated was 66 years old, female, with a history of type 2 diabetes mellitus, exogenous obesity, grade 4 fatty liver disease. He was admitted, for a diagnosis of COVID-19, presented a digestive picture due to dyspepsia, nausea, occasional diarrhea , for which prokinetics and anti-emetics were indicated. In addition, he presented high concentrations of ferritin, above 1500 ng / mL, and presented an increase in the erythrocyte sedimentation rate with a fall in hemoglobin. Due to this clinical picture, the specialists indicated treatment with the pharmaceutical composition that comprises the peptide identified as SEQ ID NO: 1, at a dose of 1 mg, every 12 hours.
Sorprendentemente, al cabo de 96 horas de tratamiento los valores de ferritina se redujeron a una concentración de 350 ng/mL. El tratamiento con la composición farmacéutica que comprende el péptido identificado como SEQ ID NO: 1 continúo durante 72 horas más. Al completar el tratamiento, la concentración de ferritina se normalizó, y el paciente no presentaba ninguna sintomatología. Surprisingly, after 96 hours of treatment the ferritin values were reduced to a concentration of 350 ng / mL. Treatment with the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1 continued for a further 72 hours. Upon completion of the treatment, the ferritin concentration normalized, and the patient did not present any symptoms.
Estos resultados indican que el tratamiento con la composición farmacéutica que comprende el péptido identificado como SEQ ID NO: 1 , es capaz de reducir la concentración de ferritina, cuando esta aumenta debido a una infección viral. Ejemplo 6. Tratamiento de paciente con pericarditis de etiología viral con la composición farmacéutica que comprende el péptido identificado como SEQ ID NO: 1. El paciente tratado tenía 48 años de edad, fue confirmado con Covid-19, e ingresado en el Hospital Militar de Holguín. Al cabo de dos días de ingreso, el paciente presentó fiebre de 38 QC y dos deposiciones diarreicas, y se le diagnostica pericarditis secundaria a miocarditis, de etiología viral (Covid-19), sin repercusión hemodinámica. El paciente no presentó distrés respiratorio. Se inició el tratamiento con la composición farmacéutica que comprende el péptido identificado como SEQ ID NO: 1 , a una dosis de 1 mg cada 12 horas. These results indicate that treatment with the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1, is capable of reducing the concentration of ferritin, when it increases due to a viral infection. Example 6. Treatment of a patient with pericarditis of viral etiology with the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1. The treated patient was 48 years old, confirmed with Covid-19, and admitted to the Holguín Military Hospital. After two days of admission, the patient presented a fever of 38C and two diarrheal stools, and was diagnosed with pericarditis secondary to myocarditis, of viral etiology (Covid-19), without hemodynamic repercussions. The patient did not present respiratory distress. Treatment was started with the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1, at a dose of 1 mg every 12 hours.
Sorprendentemente, al cabo de 24 horas de tratamiento el electrocardiograma presentó mejoría, y a las 96 horas de tratamiento el paciente se encontraba asintomático, con mejoría clínica significativa. Estos resultados confirmaron que la composición farmacéutica que comprende el péptido identificado como SEQ ID NO:1 disminuye la inflamación que tiene lugar en patologías como la pericarditis secundaria a miocarditis, sin repercusión hemodinámica, de etiología viral. Surprisingly, after 24 hours of treatment, the electrocardiogram showed improvement, and at 96 hours of treatment the patient was asymptomatic, with significant clinical improvement. These results confirmed that the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1 reduces the inflammation that occurs in pathologies such as pericarditis secondary to myocarditis, without hemodynamic repercussions, of viral etiology.
Ejemplo 7. Tratamiento de paciente con encefalopatía hepática por Covid-19 con la composición farmacéutica que comprende el péptido identificado como SEQ ID NO: 1. Example 7. Treatment of a patient with Covid-19 hepatic encephalopathy with the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1.
Paciente del sexo femenino, 55 años de edad, raza blanca, con antecedentes patológicos de hipertensión arterial. Dicha paciente ingresó en el hospital Luis Díaz Soto, en sala de aislamiento, servicio de medicina interna, al ser positiva al coronavirus que provoca la enfermedad Covid-19. Por ello, se le comenzó el tratamiento para dicha enfermedad, según el protocolo establecido en el país. Durante el ingreso estuvo asintomática hasta el décimo día. Dicho día presentó episodios de ansiedad o nerviosismo, asociado a cifras tensionales elevadas (160/90 mm Hg). Fue valorada por especialistas en psiquiatría, y se le realizaron exámenes de laboratorio complementarios. El paciente no presentó distrés respiratorio. El psiquiatra realizó el diagnostico de síndrome ansioso, con elementos de un síndrome psicopático. Female patient, 55 years old, Caucasian, with a pathological history of arterial hypertension. Said patient was admitted to the Luis Díaz Soto hospital, in the isolation room, internal medicine service, being positive for the coronavirus that causes the Covid-19 disease. Therefore, treatment for this disease was started, according to the protocol established in the country. During admission, she was asymptomatic until the tenth day. That day he presented episodes of anxiety or nervousness, associated with high blood pressure (160/90 mm Hg). She was evaluated by specialists in psychiatry, and complementary laboratory tests were performed. The patient did not present respiratory distress. The psychiatrist made the diagnosis of anxiety syndrome, with elements of a psychopathic syndrome.
El paciente empeoró su cuadro clínico al día 11 , presentó vómitos, cifras tensionales elevadas, tendencia a la taquicardia, sin relajación de esfínteres, por lo que se traslada para la unidad de cuidados intensivos. La hemogasometría evidenció la existencia de hiponatremia grave (concentración de Na 105 mmol/L). La Tomografía Axial Computarizada no presentó alteraciones craneoencefálicas, y los exámenes complementarios indicaron las enzimas hepáticas marcadamente elevadas, así como una leucocitosis. A partir de estos resultados el paciente fue diagnosticado con un cuadro clínico correspondiente a una encefalopatía hepática. The patient worsened his clinical picture on day 11, presented vomiting, high blood pressure figures, a tendency to tachycardia, without sphincter relaxation, so he was transferred to the intensive care unit. Hemogasometry showed the existence of severe hyponatraemia (Na concentration 105 mmol / L). The Computed Axial Tomography did not show cranioencephalic alterations, and the complementary tests indicated markedly elevated liver enzymes, thus like a leukocytosis. Based on these results, the patient was diagnosed with a clinical picture corresponding to hepatic encephalopathy.
Al paciente se le suspendieron todos los medicamentos hepatotóxicos, se le realizaron los cambios en la dieta, se continuó la reposición paulatina del sodio, y se inició el tratamiento con la composición farmacéutica que comprende el péptido identificado como SEQ ID NO:1 , a razón de 1 mg cada 12 horas, por vía endovenosa. The patient was suspended all the hepatotoxic medications, changes were made to the diet, the gradual replacement of sodium was continued, and the treatment with the pharmaceutical composition that comprises the peptide identified as SEQ ID NO: 1 was started, at a rate of 1 mg every 12 hours, intravenously.
Se le realizó un seguimiento clínico diario con los exámenes complementarios de laboratorio. Sorprendentemente, después de 72 horas de tratamiento con la composición farmacéutica que comprende el péptido identificado como SEQ ID NO: 1 , la evolución fue favorable, con el descenso de las enzimas hepáticas, descenso progresivo de la creatina quinasa y de los azoados en sangre, así como la normalización de la concentración del sodio. A las 96 horas de tratamiento, el cuadro correspondiente a una encefalopatía hepática se revirtió completamente, el tratamiento con la composición farmacéutica que comprende el péptido se mantuvo por tres días más. Estos resultados confirmaron que la administración de la composición farmacéutica que comprende el péptido identificado como SEQ ID NO: 1 disminuye la inflamación que tiene lugar en patologías como la encefalopatía hepática. Ejemplo 8. Tratamiento de paciente con dengue con la composición farmacéutica que comprende el péptido identificado como SEQ ID NO: 1. A daily clinical follow-up was carried out with complementary laboratory tests. Surprisingly, after 72 hours of treatment with the pharmaceutical composition that comprises the peptide identified as SEQ ID NO: 1, the evolution was favorable, with a decrease in liver enzymes, a progressive decrease in creatine kinase and nitrogen compounds in the blood. as well as the normalization of the sodium concentration. At 96 hours of treatment, the picture corresponding to a hepatic encephalopathy was completely reversed, the treatment with the pharmaceutical composition comprising the peptide was maintained for three more days. These results confirmed that the administration of the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1 reduces the inflammation that occurs in pathologies such as hepatic encephalopathy. Example 8. Treatment of dengue patient with the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1.
Paciente del sexo femenino, de 19 años, que acudió al hospital Luis Díaz Soto, por presentar fiebre de 38,5 QC, por más de 72 horas, y cefalea intensa holocraneal. Además, presentaba dolor retroocular asociado a discreta inyección conjuntival. A partir de dichos síntomas, se decide su ingreso hospitalario por sospecha de dengue. The female patient, aged 19, who came to the hospital Luis Diaz Soto, with fever of 38.5 Q C for more than 72 hours, severe headache holocraneal. In addition, she presented retroocular pain associated with a discrete conjunctival injection. Based on these symptoms, a decision is made to admit him to hospital due to suspected dengue.
Al examen físico de dicha paciente, se constató hipertermia de 39,9 QC, tensión arterial de 90/60 mm Hg, taquicardia y discreta frialdad distal. A la inspección de la piel, el médico detectó petequias en la región de los antebrazos y el abdomen, con prueba de lazo positiva. Se trató terapéuticamente con soporte de volumen a 30 mL/Kg de peso corporal, por 24 horas. The physical examination of this patient revealed hyperthermia of 39.9 QC , blood pressure of 90/60 mm Hg, tachycardia and mild distal coldness. Upon inspection of the skin, the doctor detected petechiae in the region of the forearms and abdomen, with a positive loop test. He was treated therapeutically with volume support at 30 mL / Kg of body weight, for 24 hours.
Al cabo de las 24 horas de ingreso, el paciente comenzó con dolor abdominal y vómitos. Los exámenes complementarios indicaron leucopenia (2,9 x 109 leucocitos totales), eritrosedimentación de 95 mm/h, proteína C reactiva 150 mg/mL, Lactato Deshidrogenasa 918 U/L, Transaminasa Glutámico Oxalacética 93 Ul/L, Transaminasa Glutámico Pirúvica 58 Ul/L. El ultrasonido abdominal indicó presencia de líquido libre en los cuatro ángulos, de moderada intensidad. La vesícula presentó paredes engrosadas y edema perivesicular, sin litiasis. Se le aplicaron al paciente medidas terapéuticas de sostén, que incluyeron soluciones cristaloides a razón de 35 - 45 mL/Kg de peso, para 24 horas. After 24 hours of admission, the patient began with abdominal pain and vomiting. Complementary tests indicated leukopenia (2.9 x 10 9 total leukocytes), erythrocyte sedimentation rate of 95 mm / h, C-reactive protein 150 mg / mL, Lactate Dehydrogenase 918 U / L, Glutamic Oxaloacetic Transaminase 93 Ul / L, Glutamic Pyruvic Transaminase 58 Ul / L. Abdominal ultrasound indicated the presence of free fluid in all four angles, of moderate intensity. The gallbladder presented thickened walls and perivesicular edema, without lithiasis. Supportive therapeutic measures were applied to the patient, which included crystalloid solutions at a rate of 35-45 mL / kg of weight, for 24 hours.
La tórpida evolución del paciente y la escasa respuesta a las medidas terapéuticas basadas en la reposición de líquidos, hizo sospechar al equipo médico que el paciente desarrolló el síndrome de activación de macrófagos, secundario a la infección por el virus que causa el dengue. Esto se comprobó por los valores de los parámetros de laboratorio asociados a dicho síndrome, ferritina: 1893 mg/L; fibrinógeno: 2,31 g/L; proteína C reactiva: 150 mg/mL; eritrosedimentación: 53 mm/h. The torpid evolution of the patient and the poor response to therapeutic measures based on fluid replacement, made the medical team suspect that the patient developed macrophage activation syndrome, secondary to infection by the virus that causes dengue. This was verified by the values of the laboratory parameters associated with this syndrome, ferritin: 1893 mg / L; fibrinogen: 2.31 g / L; C-reactive protein: 150 mg / mL; erythrocyte sedimentation: 53 mm / h.
A partir de estos resultados, se trasladó el paciente a la unidad de cuidados intensivos, y se le administró la composición farmacéutica que comprende el péptido identificado como SEQ ID NO:1 , a razón de 1 mg cada 12 horas, por vía endovenosa. La administración de dicha composición tuvo el objetivo de modular la respuesta inmunológica, y controlar así la tormenta de citocinas, que según el cuadro clínico y los parámetros de laboratorio se desencadenó en este paciente. Al cabo de las 48 horas de tratamiento, se observó una mejoría ostensible en el paciente, ya que desaparecieron el dolor abdominal, la hipotensión arterial y la frialdad distal. El paciente inició la ingestión de alimentos por vía oral. Al cabo de las 72 horas, continuó la mejoría clínica y radiológica, a partir de lo cual se redujo la frecuencia de administración de la composición farmacéutica que comprende el péptido a 1 mg cada 24 horas. Based on these results, the patient was transferred to the intensive care unit, and the pharmaceutical composition comprising the peptide identified as SEQ ID NO: 1 was administered, at a rate of 1 mg every 12 hours, intravenously. The administration of said composition had the objective of modulating the immune response, and thus controlling the cytokine storm, which, according to the clinical picture and laboratory parameters, was triggered in this patient. After 48 hours of treatment, a noticeable improvement was observed in the patient, since abdominal pain, arterial hypotension and distal coldness disappeared. The patient began taking food by mouth. After 72 hours, clinical and radiological improvement continued, from which the frequency of administration of the pharmaceutical composition comprising the peptide was reduced to 1 mg every 24 hours.
Al cabo del séptimo día de tratamiento, el paciente se encontraba asintomático, desde el punto de vista clínico y radiológico. Se confirmó la infección por el virus dengue, al resultar IgM positivo en la prueba serológica. Todos los parámetros de laboratorio se normalizaron, y se decidió el egreso del paciente. Estos resultados confirmaron los esbozados en los ejemplos anteriores, se confirma que la composición farmacéutica que comprende el péptido identificado como SEQ ID NO:1 controla la tormenta de citocinas, que puede estar vinculada al síndrome hemofagocítico que puede estar asociado a diversas patologías, como es el caso de pacientes con dengue que transitan a un estadio grave de la enfermedad. Ejemplo 9. Efectos de la concentración de la sacarosa en la potenciación farmacológica del péptido de SEQ ID NO:1 para la reducción de la inflamación. At the end of the seventh day of treatment, the patient was clinically and radiologically asymptomatic. Dengue virus infection was confirmed, as IgM was positive in the serological test. All laboratory parameters were normalized, and the patient was discharged. These results confirmed those outlined in the previous examples, it is confirmed that the pharmaceutical composition that comprises the peptide identified as SEQ ID NO: 1 controls the cytokine storm, which can be linked to the hemophagocytic syndrome that can be associated with various pathologies, such as the case of patients with dengue who transition to a severe stage of the disease. Example 9. Effects of the sucrose concentration on the pharmacological potentiation of the peptide of SEQ ID NO: 1 for the reduction of inflammation.
Con el objetivo de usar el péptido identificado como SEQ ID NO:1 , como inductor de mecanismos moleculares que producen la reducción de la hiperinflamación en pacientes con COVID-19 y otras patologías, estudiamos la influencia del azúcar sacarosa en la composición, en un rango de concentraciones, para seleccionar la concentración óptima de este azúcar, asociada a la inducción de células T reguladoras, cuando la formulación es aplicada por vía intravenosa. In order to use the peptide identified as SEQ ID NO: 1, as an inducer of molecular mechanisms that reduce hyperinflammation in patients with COVID-19 and other pathologies, we studied the influence of sugar sucrose on the composition, in a range of concentrations, to select the optimal concentration of this sugar, associated with the induction of regulatory T cells, when the formulation is applied intravenously.
Para la preparación de la composición se utilizaron de 1 ,8mg/mL - 3,5mg/mL del péptido identificado como SEQ ID NO:1 y se disolvió en tampón acetato de sodio, pH 4; a lo que se le adicionó el azúcar sacarosa en diferentes concentraciones, como se describe posteriormente. For the preparation of the composition, 1.8mg / mL - 3.5mg / mL of the peptide identified as SEQ ID NO: 1 were used and it was dissolved in sodium acetate buffer, pH 4; to which sucrose sugar was added in different concentrations, as described later.
Ratones machos de la línea BALB/c se separaron aleatoriamente en tres grupos de 10 cada uno. Los ratones separados por grupo, fueron inoculados por vía intravenosa, de la siguiente forma: Male mice of the BALB / c line were randomly separated into three groups of 10 each. The mice separated by group were inoculated intravenously, as follows:
-Grupo 1 : composición farmacéutica del péptido identificado como SEQ ID NO: 1 , donde la sacarosa se adicionó a una concentración de 10 mg/mL. -Grupo 2: composición farmacéutica del péptido identificado como SEQ ID NO: 1 , donde la sacarosa se adicionó a una concentración de 30 mg/mL. -Grupo 3: composición farmacéutica del péptido identificado como SEQ ID-Group 1: pharmaceutical composition of the peptide identified as SEQ ID NO: 1, where sucrose was added at a concentration of 10 mg / mL. -Group 2: pharmaceutical composition of the peptide identified as SEQ ID NO: 1, where sucrose was added at a concentration of 30 mg / mL. -Group 3: pharmaceutical composition of the peptide identified as SEQ ID
NO: 1 , donde la sacarosa se adicionó a una concentración de 80 mg/mL. Cuatro días después, los ratones recibieron una segunda dosis en idénticas condiciones. Los 10 animales por grupo se sacrificaron el día 8 después de recibir la primera inoculación. Las células del bazo de los ratones se preincubaron con el AcM 2.4G2 ( eBiosciences , EUA), para bloquear las uniones no específicas a los receptores FC. Seguidamente, las células se marcaron en PBS conteniendo tres porcentaje de suero de ternero y cantidades saturantes de los siguientes AcM ( eBiosciences , EUA): APC-Cy7-conjugado anti-CD4 (clon RM4-5) y APC-conjugado anti-CD25 (PC61). El factor transcripcional Foxp3 se evaluó a través del uso de un AcM anti-Foxp3 conjugado a PE (FJK-16s; eBioscience, EUA), siguiendo las recomendaciones de los fabricantes. Un AcM con isotipo irrelevante (PE-Cy5 lgG2a de rata; eBioscience, EUA) se utilizó como control. La región correspondiente a los linfocitos se enmarcó de acuerdo a las características de distribución de estos en los histogramas de citometría de flujo en un citómetro LSR-II™. Los análisis se efectuaron empleando el programa FlowJo® (Tree Star). NO: 1, where sucrose was added at a concentration of 80 mg / mL. Four days later, the mice received a second dose under identical conditions. The 10 animals per group were sacrificed on day 8 after receiving the first inoculation. The spleen cells of the mice were preincubated with Mab 2.4G2 (eBiosciences, USA), to block non-specific binding to FC receptors. Next, the cells were labeled in PBS containing three percent calf serum and saturating amounts of the following mAbs (eBiosciences, USA): APC-Cy7-anti-CD4 conjugate (clone RM4-5) and APC-anti-CD25 conjugate ( PC61). The Foxp3 transcriptional factor was evaluated through the use of an anti-Foxp3 mAb conjugated to PE (FJK-16s; eBioscience, USA), following the manufacturers' recommendations. An isotype-irrelevant mAb (rat PE-Cy5 IgG2a; eBioscience, USA) was used as a control. The region corresponding to lymphocytes was framed according to their distribution characteristics in the Flow cytometric histograms on an LSR-II ™ cytometer. The analyzes were carried out using the FlowJo® program (Tree Star).
Como se aprecia en esta Figura 1A los ratones inoculados con la formulación conteniendo el péptido identificado como SEQ ID NO: 1 y la sacarosa a una concentración de 30 mg/mL, aumentaron de forma significativa el porcentaje de las células T reguladoras, lo que demuestra un efecto sinérgico entre esos componentes de la composición. Con el objetivo de precisar si la sacarosa a esta concentración es determinante para inducir la frecuencia de las células T reguladoras, realizamos un experimento en condiciones muy similares al anterior, pero estrechando el rango de concentración de dicho azúcar. As can be seen in this Figure 1A, the mice inoculated with the formulation containing the peptide identified as SEQ ID NO: 1 and sucrose at a concentration of 30 mg / mL, significantly increased the percentage of regulatory T cells, which shows a synergistic effect between those components of the composition. In order to determine whether sucrose at this concentration is decisive to induce the frequency of regulatory T cells, we carried out an experiment under conditions very similar to the previous one, but narrowing the concentration range of said sugar.
Los ratones se separaron aleatoriamente en tres grupos de 10 ratones cada uno. Los ratones separados por grupo, fueron inoculados por vía intravenosa, de la siguiente forma: The mice were randomly separated into three groups of 10 mice each. The mice separated by group were inoculated intravenously, as follows:
- Grupo 1 : composición farmacéutica del péptido identificado como SEQ ID NO: 1 , donde la sacarosa se adicionó a una concentración de 20 mg/mL.- Group 1: pharmaceutical composition of the peptide identified as SEQ ID NO: 1, where sucrose was added at a concentration of 20 mg / mL.
- Grupo 2: composición farmacéutica del péptido identificado como SEQ ID- Group 2: pharmaceutical composition of the peptide identified as SEQ ID
NO: 1 , donde la sacarosa se adicionó a una concentración de 30 mg/mL.NO: 1, where sucrose was added at a concentration of 30 mg / mL.
- Grupo 3: composición farmacéutica del péptido identificado como SEQ ID- Group 3: pharmaceutical composition of the peptide identified as SEQ ID
NO: 1 , donde la sacarosa se adicionó a una concentración de 40 mg/mL. Sorprendentemente, los ratones tratados con estas concentraciones de sacarosa entre los 20-40 mg/mL mostraron un aumento significativo del porcentaje de las células T reguladoras respecto a los grupos tratados con 10-80 mg/mL del experimento anterior, observándose un efecto sinérgico entre los componentes de la composición, demostrando un mayor efecto en los grupos de ratones tratados con la composición farmacéutica del péptido identificado como SEQ ID NO:1 , donde la sacarosa se adicionó en las concentraciones de 20 y 30 mg/mL (Figura 1 B). NO: 1, where sucrose was added at a concentration of 40 mg / mL. Surprisingly, the mice treated with these sucrose concentrations between 20-40 mg / mL showed a significant increase in the percentage of regulatory T cells compared to the groups treated with 10-80 mg / mL in the previous experiment, observing a synergistic effect between the components of the composition, demonstrating a greater effect in the groups of mice treated with the pharmaceutical composition of the peptide identified as SEQ ID NO: 1, where sucrose was added in concentrations of 20 and 30 mg / mL (Figure 1 B) .
Estos resultados nos permiten concluir que la adición de sacarosa en concentraciones en el rango de 20-30 mg/mL, es un factor determinante para la inducción de las células T reguladoras, las cuales controlan la magnitud de la inflamación, así como que esta formulación aplicada por vía intravenosa tiene capacidad para controlar las patologías caracterizadas por la hiperinflamación. These results allow us to conclude that the addition of sucrose in concentrations in the range of 20-30 mg / mL is a determining factor for the induction of regulatory T cells, which control the magnitude of inflammation, as well as that this formulation applied intravenously, it has the capacity to control pathologies characterized by hyperinflammation.

Claims

REIVINDICACIONES 1. Composición farmacéutica para el tratamiento y la prevención del síndrome de la tormenta de citocinas que comprende el péptido identificado como SEQ ID No. 1 y un excipiente farmacéuticamente aceptable CLAIMS 1. Pharmaceutical composition for the treatment and prevention of cytokine storm syndrome comprising the peptide identified as SEQ ID No. 1 and a pharmaceutically acceptable excipient
2. Composición farmacéutica de la reivindicación 1 caracterizada porque comprende el péptido identificado como SEQ ID NO:1 en un rango de concentraciones entre 1 ,8 mg/mL - 3,6 mg/mL y sacarosa en una de concentración entre 20-30 mg/mL. 2. Pharmaceutical composition of claim 1 characterized in that it comprises the peptide identified as SEQ ID NO: 1 in a concentration range between 1.8 mg / mL - 3.6 mg / mL and sucrose in a concentration between 20-30 mg / mL.
3. La composición de la reivindicación 1 caracterizada porque se aplica por la vía endovenosa. 3. The composition of claim 1 characterized in that it is applied intravenously.
4. Uso de una composición farmacéutica que comprende el péptido identificado como SEQ ID No. 1 para la fabricación de un medicamento para el tratamiento y la prevención del síndrome de la tormenta de citocinas. 4. Use of a pharmaceutical composition comprising the peptide identified as SEQ ID No. 1 for the manufacture of a medicament for the treatment and prevention of cytokine storm syndrome.
5. El uso de la reivindicación 4 donde el síndrome de la tormenta de citocinas se manifiesta como síndrome de distrés respiratorio agudo, síndrome de activación de macrófagos, sepsis respiratoria en adultos o neonatos, síndrome de respuesta inflamatoria sistémica, tromboembolismo venoso o arterial, elevación aguda de los niveles de ferritina, pericarditis severa, elevación aguda de las transaminasas hepáticas que causan encefalopatía hepática o enfermedad asociada a las terapias con células T que poseen receptor de antígeno quimérico. 5. The use of claim 4 wherein the cytokine storm syndrome manifests as acute respiratory distress syndrome, macrophage activation syndrome, respiratory sepsis in adults or neonates, systemic inflammatory response syndrome, venous or arterial thromboembolism, elevation acute ferritin levels, severe pericarditis, acute elevation of liver transaminases causing hepatic encephalopathy, or disease associated with chimeric antigen receptor T-cell therapies.
6. El uso de la reivindicación 4 donde la composición se administra por vía intravenosa. 6. The use of claim 4 wherein the composition is administered intravenously.
7. El uso de la reivindicación 4 donde el síndrome de la tormenta de citocinas es causado por una enfermedad infecciosa. 7. The use of claim 4 wherein the cytokine storm syndrome is caused by an infectious disease.
8. El uso de la reivindicación 7 donde la enfermedad infecciosa es la Covid-19 o el dengue. 8. The use of claim 7 where the infectious disease is Covid-19 or dengue.
9. Método para el tratamiento o prevención del síndrome de la tormenta de citocinas que comprende la administración de una cantidad farmacéuticamente efectiva de una composición farmacéutica que comprende el péptido identificado como SEQ ID No. 1. 9. Method for the treatment or prevention of cytokine storm syndrome comprising the administration of a pharmaceutically effective amount of a pharmaceutical composition comprising the peptide identified as SEQ ID No. 1.
10. El método de la reivindicación 9 donde el síndrome de la tormenta de citocinas se manifiesta como síndrome de distrés respiratorio agudo, síndrome de activación de macrófagos, sepsis respiratoria en adultos o neonatos, síndrome de respuesta inflamatoria sistémica, tromboembolismo venoso o arterial, elevación aguda de los niveles de ferritina, pericarditis severa, elevación aguda de las transaminasas hepáticas que causan encefalopatía hepática o enfermedad asociada a las terapias con células T que poseen receptor de antígeno quimérico. The method of claim 9 wherein the cytokine storm syndrome manifests as acute respiratory distress syndrome, macrophage activation syndrome, respiratory sepsis in adults or neonates, systemic inflammatory response syndrome, venous or arterial thromboembolism, elevation acute ferritin levels, severe pericarditis, acute elevation of liver transaminases causing hepatic encephalopathy, or disease associated with chimeric antigen receptor T-cell therapies.
11. El método de la reivindicación 9 donde la composición se administra por vía intravenosa. The method of claim 9 wherein the composition is administered intravenously.
12. El método de la reivindicación 9 donde el síndrome de la tormenta de citocinas es causado por una enfermedad infecciosa. 12. The method of claim 9 wherein the cytokine storm syndrome is caused by an infectious disease.
13. El método de la reivindicación 12 donde la enfermedad infecciosa es la Covid- 19 o el dengue. 13. The method of claim 12 where the infectious disease is Covid-19 or dengue.
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CU24508B1 (en) * 2017-12-29 2021-04-07 Centro De Ingenieria Genetica Y Biotecnologia Biocubafarma PHARMACEUTICAL COMPOSITION INCLUDING APL TYPE PEPTIDE

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